Compositions and Methods for Tissue Repair with Extracellular Matrices

ABSTRACT

Described herein are compositions comprising decellularized extracellular matrix derived from skeletal muscle or other suitable tissue, and therapeutic uses thereof. Methods for treating, repairing or regenerating defective, diseased, damage, ischemic, ulcer cells, tissues or organs in a subject preferably a human, with diseases, such as PAD and CLI, using a decellularized extracellular matrix of the invention are provided. Methods of preparing culture surfaces and culturing cells with absorbed decellularized extracellular matrix are provided.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2012/054058 which claims priority to U.S. Provisional ApplicationNo. 61/531,931, filed Sep. 7, 2011, the entire contents of which areincorporated by reference herewith.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant No. OD004309awarded by National Institutes of Health (NIH). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Peripheral artery disease (PAD) is a common condition in which bloodflow is reduced to the limbs, typically the leg and feet (Manzi et al.,2011; Stansby and Williams, 2011), and if untreated, may progress to thestage of critical limb ischemia (CLI), which is the most advanced formof PAD often leading to amputation of the limb and potential mortality(Chan and Cheng, 2011; Dattilo and Casserly, 2011). Similar tomyocardial infarction (MI), which is also a result of atherosclerosis,PAD has a large population of affected individuals, with an estimated 27million suffering from PAD in Europe and North America (Belch et al.,2003). Despite recent medical advances and the advent of tissueengineering strategies, revascularization through surgical orendovascular intervention, remains the only treatment. This is furthercomplicated by the fact that approximately 40% of patients with criticallimb ischemia (CLI) are not candidates for revascularization procedures(Sprengers et al., 2008), and that revascularization has limited benefitwhen the PAD is diffuse or below the knee. This corresponds toapproximately 120,000 leg amputations in the US and 100,000 in theEuropean Union each year (Lawall et al., 2010). There is therefore apressing need for the development of new therapies for treating PAD andCLI.

Alternative therapies for PAD and CLI have largely mirrored the attemptsfor MI and heart failure, including cell transplantation and angiogenicgrowth factor therapy (Fadini et al., 2010; Menasche, 2010; Tongers etal., 2008). The goal of these therapies has been to increasevascularization of the ischemic limb so that perfusion is sufficient forwound healing to occur, and to resolve pain at rest, thereby alsopreventing limb amputations. Biomaterial based strategies have recentlybeen explored. Currently, only poly(d,l-lactide-co-glycolide) (PLGA),collagen-fibronectin, alginate, gelatin, fibrin, and peptide amphiphileshave been examined (Doi et al., 2007; Jay et al., 2008; Kong et al.,2008; Layman et al., 2007; Lee et al., 2010; Ruvinov et al., 2010; Silvaand Mooney, 2007; Webber et al., 2011). PLGA microspheres in alginatehydrogels have been utilized to deliver vascular endothelial growthfactor (VEGF) (Lee et al., 2010), and alginate hydrogels have beenexplored for delivery of hepatocyte growth factor (HGF) (Ruvinov et al.,2010), VEGF (Silva and Mooney, 2007), and pDNA encoding for VEGF (Konget al., 2008). Alginate microspheres within an injectable collagenmatrix have also been used to deliver stromal cell-derived factor-1(SDF-1) (Kuraitis et al., 2011), while VEGF loaded alginatemicroparticles in a collagen-fibronectin scaffold were used to deliverendothelial cells (Jay et al., 2008). Gelatin hydrogels have beenemployed to deliver basic fibroblast growth factor (bFGF) (Doi et al.,2007; Layman et al., 2007), and fibrin scaffolds were utilized todeliver bFGF and granulocyte-colony stimulating factor (G-CSF) alongwith bone marrow cells. More recently, VEGF-mimetic peptides weredelivered using assembling peptide amphiphiles (Webber et al., 2011).Each of these studies demonstrated enhanced cell transplantation and/orenhanced growth factor/gene delivery with resulting enhancements inneovascularization.

While each of the materials currently explored for treating PAD havebeen used extensively as tissue engineering scaffolds, they do not mimicthe extracellular microenvironment of the skeletal muscle they areintended to treat.

Recently, several clinical trials using cell therapy have demonstratedpromising results (Alev et al., 2011; Gupta and Losordo, 2011; Kawamotoet al., 2009; Menasche, 2010), but there are still many questions aboutwhich therapeutic cell type to use, quantity of cells, and best route todeliver the cells, as well as a significant problem with poor cellretention and survival (Menasché, 2010). Biomaterial scaffolds have morerecently been explored to enhance cell survival by providing a temporarymimic of the extracellular matrix (ECM) (Kawamoto et al., 2009; Laymanet al., 2011). Biomaterials have also been used for delivery of growthfactors or their mimics in animal models of PAD and CLI (Kawamoto etal., 2009; Layman et al., 2011; Ruvinov et al., 2010; Webber et al.,2011). However, these scaffolds are composed of fibrin (Layman et al.,2011), collagen-based matrix (Kuraitis et al., 2011), gelatin (Layman etal., 2007), self-assembling peptide amphiphiles (Webber et al., 2011) oralginate (Ruvinov et al., 2010), which may not provide the properbiomimetic environment for the ischemic skeletal muscle in theseconditions. Moreover, no potential therapies employing only abiomaterial have been explored to date. An acellular, biomaterial onlyapproach may reach the clinic sooner since it has the potential to be anoff-the-shelf treatment and does not have the added complications thatcells bring, including appropriate source, the need for expansion, orpotential disease transmission. Furthermore, a biomaterial that promotesneovascularization and tissue repair on its own would obviate the needfor exogenous growth factors, and the difficulties and expenseassociated with such a combination product.

The extracellular matrix (ECM) of each tissue contains similarcomponents; however, each individual tissue is composed of a uniquecombination of proteins and proteoglycans (Lutolf and Hubbell, 2005;Uriel et al., 2009). Recent studies have shown that the ECM of varioustissues can be isolated through decellularization and utilized as atissue engineering scaffold (Merritt et al.; Ott et al., 2008; Singelynet al., 2009; Uygun et al., 2010; Valentin et al., 2010; Young et al.,2011). Other decellularized ECM materials have been used for a varietyof applications for tissue repair (Crapo et al., 2011; Gilbert et al.,2006). These scaffolds are known to promote cellular influx in a varietyof tissues (Numata et al., 2004; Rieder et al., 2006). Their degradationproducts have angiogenic (Li et al., 2004) and chemoattractant (Badylaket al., 2001; Beattie et al., 2008; Li et al., 2004; Zantop et al.,2006) properties, and also promote cell migration and proliferation(Reing et al., 2009). After removal of the cellular antigens, thesescaffolds are considered biocompatible, and both allogeneic andxenogeneic ECM devices have been approved by the FDA and are in clinicaluse (Badylak, 2007).

Hydrogels derived from decellularized ECMs, including myocardium(Singelyn et al., 2009), pericardium (Seif-Naraghi et al., 2010), andadipose tissue (Young et al., 2011), were recently developed whichassemble into porous and fibrous scaffolds upon injection in vivo. It isalso recently shown that the injectable hydrogel derived fromventricular ECM promoted endogenous cardiomyocyte survival and preservedcardiac function post-myocardial infarction (Singelyn et al., 2012).

Skeletal muscles are composed of bundles of highly oriented and densemuscle fibers, each a multinucleated cell derived from myoblasts. Themuscle fibers in native skeletal muscle are closely packed together inan extracellular three-dimensional matrix to form an organized tissuewith high cell density and cellular orientation to generate longitudinalcontraction. Skeletal muscle can develop scar tissue after injury whichleads to a loss of functionality. The engineering of muscle tissue invitro holds promise for the treatment of skeletal muscle defects as analternative to host muscle transfer. Tissue engineering compositionsmust be biocompatible and capable of being vascularised and innervated.

The reconstruction of skeletal muscle, which is lost by injury, tumorresection, or various myopathies, is limited by the lack of functionalsubstitutes. Surgical treatments, such as muscle transplantation andtransposition techniques, have had some success; however, there stillexists a need for alternative therapies. Tissue engineering approachesoffer potential new solutions; however, current options offer incompleteregeneration. Many naturally derived as well as synthetic materials havebeen explored as scaffolds for skeletal tissue engineering, but noneoffer a complex mimic of the native skeletal extracellular matrix, whichpossesses important cues for cell survival, differentiation, andmigration.

The extracellular matrix consists of a complex tissue-specific networkof proteins and polysaccharides, which help regulate cell growth,survival and differentiation. Despite the complex nature of native ECM,in vitro cell studies traditionally assess cell behavior on single ECMcomponent coatings, thus posing limitations on translating findings fromin vitro cell studies to the in vivo setting. Overcoming this limitationis important for cell-mediated therapies, which rely on cultured andexpanded cells retaining native cell behavior over time.

Typically, purified matrix proteins from various animal sources areadsorbed to cell culture substrates to provide a protein substrate forcell attachment and to modify cellular behavior. However, theseapproaches would not provide an accurate representation of the complexmicroenvironment. More complex coatings have been used, such as acombination of single proteins, and while these combinatorial signalshave shown to affect cell behavior, it is not as complete as in vivo.For a more natural matrix, cell-derived matrices have been used.Matrigel is a complex system; however, it is derived from mouse sarcoma,and does not mimic any natural tissue. While many components of ECM aresimilar, each tissue or organ has a unique composition, and a tissuespecific naturally derived source may prove to be a better mimic of thecell microenvironment.

A liquid form of skeletal muscle matrix was shown to promote thedifferentiation and maturation of C2C12 skeletal myoblast progenitorswhen used as a cell culture coating due to its ability to retain acomplex mixture of skeletal muscle ECM proteins, peptides, andproteoglycans (DeQuach et al., 2010). A decellularized skeletal musclescaffold has been previously explored for replacement of a muscle defect(Merritt et al.; Wolf et al., 2012), yet this intact scaffold would notbe amenable to treating certain non-skeletal muscle tissue disease, suchas the peripheral artery disease (PAD) and CLI.

As discussed above, the only current clinical treatment for PAD and CLIis endovascular or surgical revascularization (Dattilo and Casserly,2011). Surgical bypass was the established standard, but recentlyendovascular therapies such as angioplasty, atherectomy and stenting areused as less-invasive options. However, despite these therapies, CLIcontinues to carry a major risk of limb amputation, with rates that havenot changed significantly in 30 years (Tongers et al., 2008).Unfortunately, few therapies exist for treating the ischemic skeletalmuscle in these conditions. Biomaterials have been used to increase celltransplant survival as well as deliver growth factors to treat limbischemia; however, existing materials do not mimic the native skeletalmuscle microenvironment they are intended to treat. Furthermore, notherapies involving biomaterials alone have been examined.

SUMMARY OF THE INVENTION

The present invention provides biomaterials comprising extracellularmatrix (ECM) derived from skeletal muscle or other suitable tissue, andmethod of use thereof, for therapeutic treatment of ischemia, ulcer,and/or other damages in certain diseases. In certain embodiments, thepresent invention provides injectable biomaterials comprising skeletalmuscle extracellular matrix, and method of use thereof, for treatingperipheral artery disease (PAD), critical limb ischemia (CLI), andsymptoms and associated complications with these diseases, as well aspressure ulcers, venous ulcers, diabetic ulcers, chronic vascularulcers, tunneled/undermined wounds, surgical wounds (donor sites/grafts,post-Mohs surgery, post-laser surgery, podiatric, wound dehiscence),trauma wounds (abrasions, lacerations, second-degree burns and skintears), draining wounds, inflammation, Buerger disease, atherosclerosisobliterans, and thromboangiitis obliterans, and gangrene.

In certain embodiments, the present invention provides compositions andmethods comprising injecting or implanting in a subject with PAD and/orCLI in need an effective amount of a composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissue.In other embodiments, the present invention provides a method comprisinginjecting or implanting in a subject with PAD and/or CLI in need acomposition comprising decellularized extracellular matrix derived froma suitable tissue, including but not limited to, cardiac, pericardial,liver, brain, small intestine submucosa, bladder, lung, and vasculartissue. In certain embodiments, the injection or implantation of saidcomposition repairs damage to skeletal muscle tissue sustained by saidsubject. In other embodiments, the injection or implantation of saidcomposition repairs damage caused by ischemia in said subject. Thecomposition of the present invention comprising the ECM material candegrade within about one month, two months, or three months followinginjection or implantation. In certain embodiments, the injection orimplantation of said composition repairs damage to skeletal muscletissue sustained by said subject. In certain embodiments, the injectionor implantation of said composition repairs damage caused by ischemia insaid subject. Herein, said effective amount can be an amount thatincreases blood flow in the area of the injection or implantation ortreated limb of the treated subject. In some instances the effectiveamount is an amount that increases the Ankle-Brachial Index of thetreated subject. In some instances the effective amount is an amountthat increases blood flow as measured by Doppler waveform analysis,pulse volume recording, duplex arterial ultrasound study, or exerciseDoppler stress testing. In some instances, the effective amount is anamount that increases muscle mass in the area of the injection orimplantation or treated limb of the treated subject. In some instances,the effective amount is an amount that induces new vascular formation inthe area of the injection or implantation of the treated subject. Thepresent invention provides that a liquid form of skeletal muscle matrixcan assemble into a fibrous scaffold upon injection in vivo. Thematerial can also be processed into a lyophilized form that onlyrequires sterile water, PBS, or saline to resuspend prior to injection,which can provide ease of storage and use in a clinical setting. Theinjectable skeletal muscle material of the present invention promotesproliferation of vascular cells and muscle progenitors in vitro, andthat the hydrogel enhances neovascularization as well as theinfiltration of muscle progenitors and proliferating muscle cells invivo in a hindlimb ischemia model, thereby demonstrating its capabilityfor treating PAD and CLI by not only treating ischemia, but alsopromoting tissue repair. The composition can further comprise cells,drugs, proteins, or polysaccharides. In some instances, the compositionis coated on a device such as an implant. The composition can bedelivered as a liquid, and in many instances, the composition cantransition to a gel form after delivery. In certain embodiments, thecomposition is delivered as a powder.

In one aspect, the invention provides a composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissueor other suitable tissues. The composition can be injectable. Thecomposition can be formulated into a powder or particulate. In otherinstances, the composition can be formulated to be in liquid form atroom temperature, typically 20° C. to 25° C., and in gel form at atemperature greater than room temperature or greater than 35° C. In someinstances, the composition is configured to be delivered to a tissueparenterally, such as through a small gauge needle (e.g., 27 gauge orsmaller). In some instances, said composition is suitable for directimplantation into a patient. The composition can be formulated either ina dry or hydrated form to be placed on or in wounds.

In some instances the composition comprises native proteins. In someinstances the composition comprises native peptides. In some instancesthe composition comprises native glycosaminoglycans. In some instances,the composition further comprises non-naturally occurring factors thatrecruit cells into the composition, encourage growth or preventinfection. In some embodiments, the composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissueherein retains native glycosaminoglycans. In some instances, thecomposition comprises naturally occurring factors that recruit cellsinto the composition, encourage growth or prevent infection.

In some instances, the composition further comprises a population ofexogenous or autologous therapeutic cells. The cells can be stem cellsor other precursors of skeletal muscle cells or other cell types.

In some instances, the composition further comprises a therapeuticagent, and as such is configured as a drug delivery vehicle. In someinstances, the composition is configured to coat surfaces, such astissue culture plates or scaffolds, to culture skeletal muscle, skeletalmuscle cells, or other cell types relevant to skeletal muscle repair.

In an aspect, a method of producing a composition is disclosed hereinthat comprises decellularized skeletal muscle or other tissueextracellular matrix comprising: obtaining from a subject a skeletalmuscle or other suitable tissue sample having an extracellular matrixand non-extracellular matrix components; processing skeletal muscle orother tissue sample to remove the non-extracellular matrix component toobtain decellularized skeletal muscle or other tissue extracellularmatrix and extracellular proteins and polysaccharides; and sterilizingthe decellularized skeletal muscle or other tissue extracellular matrix.In some instances, said method is performed aseptically withoutsterilization. In some instances, said method further comprises the stepof lyophilizing and grinding up the decellularized skeletal muscle orother tissue extracellular matrix. In some instances, said methodfurther comprises the step of enzymatically treating, solubilizing, orsuspending the decellularized skeletal muscle or other tissueextracellular matrix. In some instances, said decellularized skeletalmuscle or other tissue extracellular matrix is digested with pepsin at alow pH.

In some instances, said method further comprises the step of suspendingand neutralizing said decellularized skeletal muscle or other tissueextracellular matrix in a solution. In some instances, said solution isa phosphate buffered solution (PBS) or saline solution which can beinjected through a high gauge needle into the desired tissue or organ.In some instances, said composition is formed into a gel at bodytemperature. In some instances, said composition further comprisescells, drugs, proteins or other therapeutic agents that can be deliveredwithin or attached to the composition before, during or after gelation.

In some instances, said solution is placed into tissue culture plates orwells, incubated at above 35° C. or about 37° C. to form into a gel thatis used for cell culture. In one aspect, the invention provides a methodof culturing cells on an adsorbed matrix comprising the steps of:providing a solution comprising decellularized extracellular matrixderived from skeletal muscle or other suitable tissues into a tissueculture device; incubating said tissue culture plates device; removingsaid solution; and culturing cells on the adsorbed matrix. In someinstances, said cells are skeletal muscle cells or other cell typesrelevant to skeletal muscle or other tissue repair.

In an aspect, a method of culturing cells on an adsorbed matrixcomprises the steps of: providing a solution comprising decellularizedextracellular matrix derived from skeletal muscle or other suitabletissues into a tissue culture device; incubating said tissue cultureplates device; removing said solution; and culturing cells on theadsorbed matrix. In some instances, said cells are skeletal myoblasts,stem cells or other cell types relevant to skeletal muscle repair.

In one aspect, the invention provides a therapeutic method for skeletalmuscle and/or other tissue (such as ischemic tissue, or ulcer tissue)repair in a subject comprising injecting or implanting a therapeuticallyeffective amount of a composition comprising decellularizedextracellular matrix derived from skeletal muscle or other suitabletissue into a subject in need thereof. In an aspect, a therapeuticmethod for skeletal muscle or other tissue repair in a subject comprisesimplanting a composition comprising decellularized extracellular matrixderived from skeletal muscle or other suitable tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates decellularization and tissue processing. FIG. 1(A)shows decellularized skeletal muscle matrix. FIG. 1(B) shows lyophilizedskeletal muscle matrix prior to milling. FIG. 1(C) shows digestedskeletal muscle matrix. FIG. 1(D) shows in vitro gel of the skeletalmuscle matrix with media on top in right well. FIG. 1(E) shows thatskeletal muscle matrix that has been digested and re-lyophilized. FIG.1(F) shows re-lyophilized skeletal muscle matrix resuspended using onlysterile water.

FIG. 2 illustrates in vitro mitogenic activity assay. FIG. 2(A) showsrat aortic smooth muscle cells and FIG. 2(B) shows that C2C12 skeletalmyoblasts were cultured using growth media with the addition of degradedskeletal muscle matrix, collagen, or pepsin. Proliferation rate wasincreased for both cell types when cultured in the presence of skeletalmuscle matrix degradation products.

FIG. 3 illustrates Rheological data. A representative trace of thestorage (G′) and loss (G″) moduli for the skeletal muscle matrix gel isshown.

FIG. 4 illustrates skeletal muscle matrix delivery and gelation in situ.FIG. 4(A) shows intramuscular injection of the skeletal muscle matrixmaterial. FIG. 4(B) shows gelation of the skeletal muscle matrix in situafter 20 minutes as seen after excision of the muscle; arrow denotes thewhite matrix. FIG. 4(C) shows DAB staining of the biotin-labeledskeletal muscle matrix that gelled within the muscle. Scale bar at 200μm.

FIG. 5 illustrates scanning electron microscopy. Micrograph of across-section of skeletal muscle matrix formed FIG. 5(A) in vitro, andFIG. 5(B) 20 minutes post-subcutaneous injection. Note the formation ofthe assembled fibers on the nano- and micro-scale. Scale bar at 100 μm.

FIG. 6 illustrates quantification of arterioles. FIG. 6(A) showscollagen and FIG. 6(B) shows skeletal muscle matrix injection regionsstained with anti-alpha-SMA (red greyscale) to determine arterioleformation. Vessels with a clear lumen are seen within the injectionregion at 5 days. Scale bar at 100 μm. Quantification of the vesseldensity at 3, 5, 7, and 14 days for vessels with a lumen FIG. 6(C)>10 μmor FIG. 6(D)>25 μm demonstrated that the skeletal muscle matrixincreased neovascularization. Vessels were, on average, larger in theskeletal muscle matrix when compared to collagen.

FIG. 7 illustrates quantification of endothelial cell recruitment. FIG.7(A) shows collagen and FIG. 7(B) shows skeletal muscle matrix injectionregions stained with isolectin (green greyscale) to assess endothelialcell infiltration at 5 days. Scale bar at 100 μm.* and dotted linedenote area of material. FIG. 7(C) shows that endothelial cellinfiltration at 3, 5, 7, and 14 days was similar across all four timepoints, but was significantly greater in the skeletal muscle matrixinjection region at 3 and 7 days post-injection.

FIG. 8 illustrates proliferating muscle cell recruitment. FIG. 8(A)shows collagen injection region and FIG. 8(B) shows skeletal musclematrix injection region at 5 days with desmin-stained cells (greengreyscale) co-labeled with Ki67 (red greyscale). Arrows denote desminand Ki67 positive cells. Scale bar at 20 μm. Insert shows positivedesmin staining of healthy skeletal muscle, scale bar at 100 μm. FIG.8(C) shows quantification of desmin-positive cells in the skeletalmuscle matrix compared to collagen normalized to area. Note that thereare significantly more desmin-positive cells in the skeletal musclematrix. FIG. 8(D) shows that, of these desmin-positive cells, a majorityof the cells are proliferating as seen by Ki67 co-labeling.

FIG. 9 illustrates muscle progenitor infiltration. MyoD positive cells(green greyscale) in FIG. 9(A) collagen and FIG. 9(B) skeletal musclematrix injection regions at 5 days. Area of injection is denoted by thedotted line. Scale bar at 20 μm. FIG. 9(C) shows graph of MyoD-positivecells normalized to the area for the injection region. The number ofMyoD-positive cells was significantly higher in the skeletal musclematrix regions at all time points.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a decellularized skeletal muscleextracellular matrix (ECM) composition, and method of use thereof, fortreating ischemia, ulcer, and other tissue damage, as well as forrestoring muscle mass and function in certain diseases. Described hereinare compositions comprising ECM derived from skeletal muscle tissue orother suitable tissues, including, but not limited to, cardiac,pericardial, liver, brain, small intestine submucosa, bladder, lung, andvascular tissue, which can be used for injection into skeletal muscletissue and/or other tissues in need of therapeutic treatment. In certainembodiments, the ECM composition of the present invention can also beused to support injured tissue or change the mechanical properties. Incertain embodiments, the ECM composition as described herein can helpregenerate defective or absent skeletal muscle and restore muscle massand function. In certain embodiments, the injection or implantation ofsaid composition repairs damage to skeletal muscle tissue sustained bysaid subject.

In certain embodiments, the present invention provides a decellularizedskeletal muscle extracellular matrix (ECM) composition, and method ofuse thereof, for treating peripheral artery disease (PAD) and criticallimb ischemia (CLI). The present invention provides that thedecellularized skeletal muscle extracellular matrix composition of thepresent invention increases arteriole and capillary density, as well asrecruited more desmin-positive and MyoD-positive cells compared tocollagen, indicating that the tissue specific decellularized skeletalmuscle extracellular matrix composition which can be formed in aninjectable hydrogel is a therapy for treating ischemia related to PAD,as well as beneficial effects on restoring muscle mass that is typicallylost in critical limb ischemia (CLI). In other embodiments, theinjection or implantation of said composition repairs damage caused byischemia in said subject. The composition of the present inventioncomprising the ECM derived from skeletal muscle or other suitabletissues as discussed herein degrades within about one month, two months,or three months following injection or implantation.

In certain embodiments, the present invention further provides adecellularized skeletal muscle extracellular matrix (ECM) composition,and method of use thereof, for treating other diseases resulting fromischemia, ulcer, muscle mass lost, or other tissue damage. Exemplarydiseases that can be treated using the decellularized skeletal muscleextracellular matrix (ECM) composition of the present invention include,but are not limited to pressure ulcers, venous ulcers, diabetic ulcers,chronic vascular ulcers, tunneled/undermined wounds, surgical wounds(donor sites/grafts, post-Mohs surgery, post-laser surgery, podiatric,wound dehiscence), trauma wounds (abrasions, lacerations, second-degreeburns and skin tears), draining wounds, inflammation, Buerger disease,atherosclerosis obliterans, and thromboangiitis obliterans, andgangrene.

The present invention further provides a method of delivering thedecellularized skeletal muscle extracellular matrix (ECM) composition ofthe present invention, with or without other therapeutic agents,including cells, into one or more injured tissues or organs damaged bycertain disease conditions or trauma. In some instances, methods ofdelivery are described wherein the skeletal muscle ECM composition ofthe present invention can be placed in contact with a defective,diseased or absent muscle tissues or other injured tissues, resulting inskeletal muscle tissue regeneration and restoration of muscle mass andfunction. Exemplary methods for delivery of a composition comprising theskeletal muscle ECM include, but are not limited to: direct instillationduring surgery; direct injection into the injured tissue or organ;indirect delivery through a catheter to the injured tissue or organ. Thecomposition can also be delivered as a liquid, gel or in a solidformulation, such as a graft or patch or associated with a cellularscaffold or a particulate. Dosages and frequency will vary dependingupon the needs of the patient and judgment of the physician.

In certain embodiments, the present invention provides a native skeletalmuscle ECM decellularization and gelation method to create an in situscaffold for cellular transplantation. An appropriate digestion andpreparation protocol has been provided herein that can createnanofibrous gels. The gel solution is capable of being parenterallydelivered into the skeletal muscle tissue or other injured tissue ororgan, thus providing an in situ gelling scaffold. Since adecellularized skeletal muscle ECM best mimics the natural skeletalmuscle environment, it improves cell survival and retention uponinjection at the site of the injured tissue, thus encouraging tissueregeneration.

The skeletal muscle ECM of the present invention can also be used torecruit cells into the injured tissue or as a drug delivery vehicle. Insome instances, the composition herein can recruit endogenous cellswithin the recipient and can coordinate the function of the newlyrecruited or added cells, allowing for cell proliferation or migrationwithin the composition. An extracellular matrix composition herein canfurther comprise one or more additional components, for example withoutlimitation: an exogenous cell, a peptide, polypeptide, or protein, avector expressing a DNA of a bioactive molecule, and other therapeuticagents such as drugs, cellular growth factors, nutrients, antibiotics orother bioactive molecules. Therefore, in certain preferred embodiments,the ECM composition can further comprise an exogenous population ofcells such as stem cells or progenitor, or skeletal muscle cellprecursors, as described below.

The skeletal muscle ECM of the present invention can be derived from thenative or natural matrix of mammalian skeletal muscle tissue. Theskeletal muscle ECM of the present invention can also be derived from ananimal or synthetic source. In some instances, the decellularizedskeletal muscle extracellular matrix is derived from native skeletalmuscle tissue selected from the group consisting of human, porcine,bovine, goat, mouse, rat, rabbit, or any other mammalian or animalskeletal muscle. In some embodiments, the biocompatible compositioncomprising the decellularized skeletal muscle extracellular matrix is inan injectable gel or solution form, and can be used for skeletal muscleor other tissue repair by transplanting or delivering cells containedtherein into the injured or desired tissue in need following a diseasecondition, or recruiting the patient's own cells into the injured ordesired tissue in need. In other instances, the biocompatible materialcomprising a decellularized skeletal ECM is, for example, a patch, anemulsion, a viscous liquid, fragments, particles, microbeads, ornanobeads.

In some instances, the invention provides biocompatible materials forculturing skeletal muscle cells or other skeletal muscle relevant cellsin research laboratories, or in a clinical setting prior totransplantation and for skeletal muscle or other tissue repair. Methodsfor manufacturing and coating a surface, such as tissue culture platesor wells, with decellularized skeletal extracellular matrix are alsoprovided. The biocompatible materials of the invention are also suitablefor implantation into a patient, whether human or animal.

The invention further provides a method of producing a biocompatiblematerial comprising the decellularized skeletal muscle extracellularmatrix of the invention. Such method comprises the steps of: (a)obtaining a skeletal muscle tissue sample having an extracellular matrixcomponent and non-extracellular matrix component; (b) processing theskeletal muscle tissue sample to remove at least a portion orsubstantially all the non-extracellular matrix component to obtaindecellularized skeletal muscle extracellular matrix; and (c) sterilizingthe decellularized skeletal muscle extracellular matrix. In certainembodiments, the skeletal muscle tissue sample is isolated from a mammalsuch as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.)or a primate (e.g., monkey and human), or an avian source (e.g.,chicken, duck, etc.). Decellularization procedures for the skeletaltissue sample are performed using one or more physical, chemical and/orbiological techniques, known in the art and as taught herein. Methods ofmaking the compositions herein can include decellularizing tissue fromany age animal or human by methods well known in the art.

For human therapy, there are many potential sources for the skeletalmuscle extracellular matrix material: human skeletal muscle (includingautologous, allogeneic, or cadaveric), porcine skeletal muscle, bovineskeletal muscle, goat skeletal muscle, mouse skeletal muscle, ratskeletal muscle, rabbit skeletal muscle, chicken skeletal muscle, andother animal sources. One donor skeletal muscle can be used to treatmany people. Non-human animals are a source of skeletal muscleextracellular matrix without the need for human donors. As a researchreagent, non-human animal sources can be utilized.

In certain embodiments, the method of processing the skeletal muscleextracellular matrix is as follows. The skeletal tissue is firstdecellularized, leaving only the extracellular matrix. Decellularizationcan be performed with a perfusion of sodium dodecyl sulfate andphosphate buffered solution, or other detergents, for example. Theskeletal muscle extracellular matrix is then lyophilized, ground up, anddigested with pepsin at a low pH, between about pH 1-6 or pH 1-4, orother matrix degrading enzymes such as matrix metalloproteinases.

To produce a gel form of the skeletal muscle extracellular matrix for invivo therapy, the solution comprising the skeletal muscle extracellularmatrix is then neutralized and brought up to the desired temperature,concentration and viscosity using PBS/saline. In certain embodiments,the ECM concentration can be 1-20 mg/mL, or 2-8 mg/mL. The solutioncomprising the skeletal muscle extracellular matrix can then be injectedthrough a high gauge needle, such as 27 gauge or higher, into theinjured tissue or any tissue in need. At body temperature, e.g., 36.8°C.±0.7° C., such solution then forms into a gel. Cells, drugs, proteins,or other therapeutic agents can also be delivered inside the skeletalmuscle ECM gel.

To produce a gel form of the skeletal muscle extracellular matrix for invitro uses, the solution comprising the skeletal muscle extracellularmatrix is neutralized and brought up to the desired concentration usingPBS/saline. In certain embodiments, the ECM concentration can be 1-20mg/mL, or 2-8 mg/mL. Such solution can then be placed onto any solidsurface such as into tissue culture plates/wells. Once placed in anincubator at 37° C. or above room temperature, the solution forms a gelthat can be used for cell culture.

The invention also provides a therapeutic method for skeletal muscle orother relevant tissue repair in a subject comprising injecting orimplanting in part or in its entirety the biocompatible skeletal muscleECM material of the invention into a patient. The invention furtherprovides a therapeutic method for treating PDA, CLI, or other defective,diseased, damaged, ischemic, ulcer, or other injured tissue or organ ina subject comprising injecting or implanting the biocompatible materialof the invention in situ.

The compositions herein can comprise a decellularized ECM derived fromskeletal muscle tissue and another component or components. In someinstances, the amount of ECM in the total composition is greater than90% or 95% or 99% of the composition by weight. In some embodiments, theECM in the total composition is greater than 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, or 80% of the composition by weight.

Decellularized extracellular matrices are prepared such that much of thebioactivity for skeletal muscle tissue regeneration is preserved.Exemplary bioactivity of the compositions herein include withoutlimitation: control or initiation of cell adhesion, cell migration, celldifferentiation, cell maturation, cell organization, cell proliferation,cell death (apoptosis), stimulation of angiogenesis, proteolyticactivity, enzymatic activity, cell motility, protein and cellmodulation, activation of transcriptional events, provision fortranslation events, inhibition of some bioactivities, for exampleinhibition of coagulation, stem cell attraction, chemotaxis, and MMP orother enzyme activity.

The compositions comprise an extracellular matrix that is substantiallydecellularized. In some instances, a decellularized matrix comprises noliving native cells with which the ECM naturally occurs. In someinstances, a substantially decellularized matrix comprises less than 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% native cells by weight.

As described herein, a composition can comprise a decellularizedskeletal muscle ECM and different tissue decellularized EMC or asynthetic or naturally occurring polymer from animal and non-animalsources (such as plants or synthetic collagens). For example, acomposition herein comprises a natural polymer such as collagen,chitosan, alginate, glycosaminoglycans, fibrin, or hyaluronic acid. Inanother example, a composition herein comprises a synthetic polymer, forexample without limitation, polyethylene glycol, poly(glycolic)acid,poly(lactic acid), poly(hydroxy acids), polydioxanone, polycaprolactone,poly(ortho esters), poly(anhydrides), polyphosphazenes, poly(aminoacids), pseudo-poly(amino acids), conductive polymers (such aspolyacetylene, polypyrrole, polyaniline), or polyurethane or theirpotential copolymers. In some instances, a composition here comprise ECMand both a natural and a synthetic polymer. A composition herein can bea multi-material by linking an ECM and another polymer material, forexample, via reaction with amines, free thiols, or short peptides thatcan be self-assembled with the ECM.

In some instances, a polymer of the composition is biocompatible andbiodegradable and/or bioabsorbable, and can be a random copolymer, blockcopolymer or blend of monomers, homopolymers, copolymers, and/orheteropolymers that contain these monomers. Exemplary biodegradable orbioabsorbable polymers include, but are not limited to: polylactides,poly-glycolides, polycarprolactone, polydioxane and their random andblock copolymers. A biodegradable and/or bioabsorbable polymer cancontain a monomer selected from the group consisting of a glycolide,lactide, dioxanone, caprolactone, trimethylene carbonate, ethyleneglycol and lysine. The biodegradable and/or bioabsorbable polymers cancontain bioabsorbable and biodegradable linear aliphatic polyesters suchas polyglycolide (PGA) and its random copolymerpoly(glycolide-co-lactide-) (PGA-co-PLA). Other examples of suitablebiocompatible polymers are polyhydroxyalkyl methacrylates includingethylmethacrylate, and hydrogels such as polyvinylpyrrolidone andpolyacrylamides. Other suitable bioabsorbable materials are biopolymerswhich include collagen, gelatin, alginic acid, chitin, chitosan, fibrin,hyaluronic acid, dextran, polyamino acids, polylysine and copolymers ofthese materials. Any combination, copolymer, polymer or blend thereof ofthe above examples is contemplated for use according to the presentinvention. Such bioabsorbable materials may be prepared by knownmethods.

Therefore, methods are described herein for preparing a compositioncomprising decellularized ECM derived from skeletal muscle tissue. Theinvention also provides ECM compositions and methods derived fromskeletal muscle tissue in an analogous process. Related compositions,devices and methods of production and use also are provided. In someinstances a composition comprises crosslinkers including, but notlimited to, common collagen crosslinkers, hyaluronic acid crosslinkers,or other protein cross-linkers with altered degradation and mechanicalproperties. The compositions which may include cells or othertherapeutic agents may be implanted into a patient, human or animal, bya number of methods. In some instances, the compositions are injected asa liquid into a desired site in the patient.

In certain embodiments, the viscosity of the composition increases whenwarmed above room temperature including physiological temperaturesapproaching about 37° C. According to one non-limiting embodiment, theECM-derived composition is an injectable solution at room temperatureand other temperatures below 35° C. In another non-limiting embodimentthe gel can be injected body temperature above about 37° C. or near bodytemperature, but gels more rapidly at increasing temperatures. A gelforms after approximately 15-20 minutes at physiological temperature of37° C. A general set of principles for preparing an ECM-derived gel isprovided along with preferred specific protocols for preparing gels inthe following Examples which are applicable and adaptable to numeroustissues including without limitation the skeletal muscle.

Commercially available ECM preparations can also be combined in themethods, devices and compositions described herein. In one embodiment,the ECM is derived from small intestinal submucosa (SIS). Commerciallyavailable preparations include, but are not limited to, SURGISIS™,SURGISIS-ES™, STRATASIS™, and STRATASIS-EST™ (Cook Urological Inc.;Indianapolis, Ind.) and GRAFTPATCH™ (Organogenesis Inc.; Canton, Mass.).In another embodiment, the ECM is derived from dermis. Commerciallyavailable preparations include, but are not limited to PELVICOL™ (soldas PERMACOL™ in Europe; Bard, Covington, Ga.), REPLIFORM™ (Microvasive;Boston, Mass.) and ALLODERM™ (LifeCell; Branchburg, N.J.).

In some instances, the solution, gel form, and adsorbed form of theskeletal muscle extracellular matrix of the invention provide all theconstituents at the similar ratios found in vivo. For therapeutictreatment, the skeletal muscle extracellular matrix of the invention canbe delivered which can allow for skeletal muscle or other relevanttissue repair or regeneration. For in vitro cell culture for skeletalmuscle cells and other relevant cells, the gel and adsorbed forms of theskeletal muscle extracellular matrix of the invention contain all ormany of the same extracellular matrix cues that the cells recognize invivo as compared to the commonly used collagen, laminin, SURECOAT(CELLUTRON, mixture of collagen and laminin), and gelatin.

The compositions herein provide a particulate, powder, emulsion, gel orsolution form of skeletal muscle extracellular matrix, and the use ofthese forms of skeletal muscle extracellular matrix for skeletal muscleor other relevant tissue repair or regeneration, for treatment ofcertain diseases, such as PDA, CLI, pressure ulcers, venous ulcers,diabetic ulcers, chronic vascular ulcers, tunneled/undermined wounds,surgical wounds (donor sites/grafts, post-Mohs surgery, post-lasersurgery, podiatric, wound dehiscence), trauma wounds (abrasions,lacerations, second-degree burns and skin tears), draining wounds,inflammation, Buerger disease, atherosclerosis obliterans, andthromboangiitis obliterans, and gangrene, or for cell culture. In oneembodiment, the skeletal muscle tissue is first decellularized, leavingonly the extracellular matrix. The matrix is then lyophilized, ground orpulverized into a fine powder, and solubilized with pepsin or otherenzymes, such as, but not limited to, matrix metalloproteases,collagenases, and trypsin.

For gel therapy, the solution is then neutralized and brought up to theappropriate concentration using PBS/saline. In one embodiment, thesolution can then be injected through a needle into the injured tissueor a tissue in need. The needle size can be without limitation 22 g, 23g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, or smaller. In oneembodiment, the needle size through which the solution is injected is 27g. Delivery can also occur through a balloon infusion catheter or othernon-needle catheter. Dosage amounts and frequency can routinely bedetermined based on the varying condition of the injured tissue andpatient profile. At body temperature, the solution can then form into agel. In yet another embodiment, gel can be crosslinked withglutaraldehye, formaldehyde, bis-NHS molecules, or other crosslinkers.

In yet another embodiment, the ECM can be combined with othertherapeutic agents, such as cells, peptides, proteins, DNA, drugs,nutrients, antibiotics, survival promoting additives, proteoglycans,and/or glycosaminolycans. In yet another embodiment, the ECM can becombined and/or crosslinked with a synthetic polymer. Examples ofsynthetic polymers include, but are not limited to: polyethyleneterephthalate fiber (DACRON™), polytetrafluoroethylene (PTFE),polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol(PEG),), poly(ethylene glycol) diacrylate (PEG diacrylate),polyethylene, polystyrene and nitinol.

In yet another embodiment, ECM solution or gel can be injected into theinjured tissue or other relevant tissue in need, alone or in combinationwith above-described components for endogenous cell ingrowth,angiogenesis, and regeneration. In yet another embodiment, the ECM orECM liquid can be sprayed on or into injured tissue or other relevanttissue in need, alone or in combination with above-described componentsfor endogenous cell ingrowth, angiogenesis, and regeneration. In yetanother embodiment, the composition can also be used alone or incombination with above-described components as a matrix to changemechanical properties of the skeletal muscle or other relevant tissuesand/or to restore muscle mass and function. In yet another embodiment,the composition can be delivered with cells alone or in combination withthe above-described components for regenerating muscle mass andfunction. In yet another embodiment, the composition can be used aloneor in combination with above-described components for increasingarteriole and capillary density, as well as recruiting more desiredcells for tissue repair and regeneration.

In one embodiment for making a soluble reagent, the solution is broughtup in a low pH, neutral pH, or physiological pH solution including butnot limited to 0.5 M, 0.1, or 0.01 M acetic acid or 0.1M HCl, PBS, orother buffering solutions to the desired concentration and then placedinto tissue culture plates/wells, coverslips, scaffolding or othersurfaces for tissue culture. After placing in an incubator at 37° C. for1 hour, or overnight at room temperature, the excess solution isremoved. After the surfaces are rinsed with PBS, cells can be culturedon the adsorbed matrix. The solution can be combined in advance withpeptides, proteins, DNA, drugs, nutrients, survival promoting additives,proteoglycans, and/or glycosaminoglycans before, during, or afterinjection/implantation.

The present invention provides enhanced cell attachment and survival onboth the therapeutic composition and adsorbed cell culturing compositionforms of the skeletal muscle extracellular matrix in vitro. The solublecell culturing reagent form of the skeletal muscle extracellular matrixinduces faster spreading, faster maturation, and/or improved survivalfor skeletal muscle cells and other relevant cells compared to standardplate coatings.

In an embodiment herein, a biomimetic ECM derived from native skeletalmuscle tissue is disclosed. In some instances, a matrix resembles the invivo skeletal muscle or other relevant tissue environment in that itcontains many or all of the native chemical cues found in naturalskeletal muscle ECM. In some instances, through crosslinking or additionor other materials, the mechanical properties of healthy adult orembryonic skeletal muscle can also be mimicked. As described herein,skeletal muscle ECM can be isolated and processed into a gel using asimple and economical process, which is amenable to scale-up forclinical translation.

In some instances, a composition as provided herein can comprise amatrix and exogenously added or recruited cells. The cells can be anyvariety of cells. In some instances, the cells are a variety of skeletalmuscle or relevant cells including, but not limited to: stem cells,progenitors, skeletal muscle precursor cells, and fibroblasts derivedfrom autologous or allogeneic sources.

The invention thus provides a use of a gel made from nativedecellularized skeletal muscle extracellular matrix to support isolatedneonatal skeletal muscle or stem cell progenitor derived skeletal musclecells in vitro and to act as an in situ gelling scaffold, providing anatural matrix to improve cell retention and muscle mass restoration. Ascaffold created from skeletal muscle ECM is well-suited for celltransplantation in the injured tissue, since it more closelyapproximates the in vivo environment compared to currently availablematerials.

A composition herein comprising skeletal muscle ECM and exogenouslyadded cells can be prepared by culturing the cells in the ECM. Inaddition, where proteins such as growth factors are added into theextracellular matrix, the proteins may be added into the composition, orthe protein molecules may be covalently or non-covalently linked to amolecule in the matrix. The covalent linking of protein to matrixmolecules can be accomplished by standard covalent protein linkingprocedures known in the art. The protein may be covalently or linked toone or more matrix molecules.

In one embodiment, when delivering a composition that comprises thedecellularized skeletal muscle ECM and exogenous cells, the cells can befrom cell sources for treating certain diseases, such as PAD or CLI,that include allogeneic, xenogeneic, or autogenic sources. Accordingly,embryonic stem cells, fetal or adult derived stem cells, inducedpluripotent stem cells, skeletal muscle progenitors, fetal and neonatalskeletal muscle cells, mesenchymal cells, parenchymal cells, epithelialcells, endothelial cells, mesothelial cells, fibroblasts, hematopoieticstem cells, bone marrow-derived progenitor cells, skeletal cells,macrophages, adipocytes, and autotransplanted expanded skeletal cellscan be delivered by a composition herein. In some instances, cellsherein can be cultured ex vivo and in the culture dish environmentdifferentiate either directly to skeletal muscle cells, or to bonemarrow cells that can become skeletal muscle cells. The cultured cellsare then transplanted into the mammal, either with the composition or incontact with the scaffold and other components.

Adult stem cells are yet another species of cell that can be part of acomposition herein. Adult stem cells are thought to work by generatingother stem cells (for example those appropriate to skeletal muscle) in anew site, or they differentiate directly to a skeletal muscle cells invivo. They may also differentiate into other lineages after introductionto organs, such as the skeletal muscle. The adult mammal providessources for adult stem cells in circulating endothelial precursor cells,bone marrow-derived cells, adipose tissue, or cells from a specificorgan. It is known that mononuclear cells isolated from bone marrowaspirate differentiate into endothelial cells in vitro and are detectedin newly formed blood vessels after intramuscular injection. Thus, useof cells from bone marrow aspirate can yield endothelial cells in vivoas a component of the composition. Other cells which can be employedwith the invention are the mesenchymal stem cells administered withactivating cytokines. Subpopulations of mesenchymal cells have beenshown to differentiate toward skeletal muscle generating cell lines whenexposed to cytokines in vitro.

Human embryonic stem cell derived skeletal muscle cells can be grown ona composition herein comprising the skeletal muscle ECM of the presentinvention. In some instances, hESC-derived skeletal muscle cells grownin the presence of a composition herein provide a more in vivo-likemorphology. In some instances, hESC-derived skeletal muscle cells grownin the presence of a composition herein provide increased markers ofmaturation.

The invention is also directed to a drug delivery system comprisingdecellularized skeletal muscle extracellular matrix for deliveringcells, drugs, molecules, or proteins into a subject for treatingdefective, diseased, damaged, ischemic, ulcer or other injured tissuesor organs. In one embodiment, the inventive biocompatible skeletalmuscle ECM material comprising the decellularized skeletal muscleextracellular matrix alone or in combination with other components isused for treating PAD, CLI, and other diseases to increase arteriole andcapillary density and restore muscle mass and function. Therefore, theinventive biocompatible ECM material can be used to transplant cells, orinjected alone to recruit native cells or other cytokines endogenoustherapeutic agents, or act as a exogenous therapeutic agent deliveryvehicle.

The composition of the invention can further comprise cells, drugs,proteins, or other biological material such as, but not limited to,erythropoietin (EPO), stem cell factor (SCF), vascular endothelialgrowth factor (VEGF), transforming growth factor (TGF), fibroblastgrowth factor (FGF), epidermal growth factor (EGF), cartilage growthfactor (CGF), nerve growth factor (NGF), keratinocyte growth factor(KGF), skeletal growth factor (SGF), osteoblast-derived growth factor(BDGF), hepatocyte growth factor (HGF), insulin-like growth factor(IGF), cytokine growth factor (CGF), stem cell factor (SCF),platelet-derived growth factor (PDGF), endothelial cell growthsupplement (EGGS), colony stimulating factor (CSF), growthdifferentiation factor (GDF), integrin modulating factor (IMF),calmodulin (CaM), thymidine kinase (TK), tumor necrosis factor (TNF),growth hormone (GH), bone morphogenic proteins (BMP), matrixmetalloproteinase (MMP), tissue inhibitor matrix metalloproteinase(TIMP), interferon, interleukins, cytokines, integrin, collagen,elastin, fibrillins, fibronectin, laminin, glycosaminoglycans,hemonectin, thrombospondin, heparan sulfate, dermantan, chondroitinsulfate (CS), hyaluronic acid (HA), vitronectin, proteoglycans,transferrin, cytotactin, tenascin, and lymphokines.

Tissue culture plates can be coated with either a soluble ligand or gelform of the extracellular matrix of the invention, or an adsorbed formof the extracellular matrix of the invention, to culture skeletal musclecells or other cell types relevant to skeletal muscle tissue or otherrelevant tissue repair. This can be used as a research reagent forgrowing these cells or as a clinical reagent for culturing the cellsprior to implantation. The extracellular matrix reagent can be combinedwith other tissue matrices and cells.

For gel reagent compositions, the solution is then neutralized andbrought up to the appropriate concentration using PBS/saline or otherbuffer, and then be placed into tissue culture plates and/or wells. Onceplaced in an incubator at 37° C., the solution forms a gel that can beused for any 2D or 3D culture substrate for cell culture. In oneembodiment, the gel composition can be crosslinked with glutaraldehye,formaldehyde, bis-NHS molecules, or other crosslinkers, or be combinedwith cells, peptides, proteins, DNA, drugs, nutrients, survivalpromoting additives, proteoglycans, and/or glycosaminolycans, orcombined and/or crosslinked with a synthetic polymer for further use.

The invention further provides an exemplary method of culturing cellsadsorbed on a decellularized skeletal muscle extracellular matrixcomprising the steps of: (a) providing a solution comprising thebiocompatible material of decellularized skeletal muscle ECM in low pHsolution or approximately neutral or physiological pH including but notlimited to, 0.5 M, or 0.01 M acetic acid or 0.1M HCl or PBS or any otherbuffered solution to a desired concentration, (b) placing said solutioninto tissue culture plates or wells, (c) incubating said tissue cultureplates or wells above room temperature such as at 37° C., for between 1hour and overnight (or at room temperature to 40° C.), (d) removingexcess solution, (e) rinsing said tissue culture plates or wells withPBS, and (f) culturing cells on the adsorbed matrix. Cells that can becultured on the adsorbed matrix comprising the skeletal muscleextracellular matrix of the invention include skeletal muscle cells orother cell types relevant to skeletal muscle repair, including stemcells and skeletal muscle cell progenitors.

Skeletal myoblasts plated on skeletal muscle matrix displayed asignificant increase in i) the number of myosin heavy chain positivemyotubes, ii) the number of nuclei per myotube and iii) myotube widthwhen compared to cells plated on traditional collagen type I coatedsubstrates. In some instances, the compositions are configured toprovide the ability to reconstitute the in vivo muscle ECM. Thecomposition may provide a tool to assess and maintain muscle and stemcell behavior in vitro similar to the native state, and may provide atool for cell-mediated therapies in vivo.

In one instance, a method of making the composition herein compriseselectrospinning. In some instances, a method herein is configured tocontrol the nanofiber size, shape, or thickness. In some instances,contractility can be induced into the composition, for example, withcells or external pacing. Contractility can create cyclic stress topromote a more natural skeletal muscle. In some instances, cell influxand angiogenesis can be induced into the composition, for example, whenthe composition comprises linked groups or embedded factors, such asangiogenic factors.

In some instances, a composition herein may contain microbeads.Microbeads can be a part of the composition or delivered by thecomposition. Exemplary microbeads can be any variety of materials, forexample, natural or synthetic. In some instances, the microbeads canhave varied degradation properties or comprise, for example, MMPinhibitors, growth factors, or small molecules.

In some instances, the composition can comprise a biological group thatcan act as an adhesive or anchor where the composition is delivered. Inone instance, a composition can be a bioadhesive, for example, for woundrepair. In some instances, a composition herein can be configured as acell adherent. For example, the composition herein can be coating ormixed with on a medical device or a biologic that does or does notcomprises cells. For example, the composition herein can be a coatingfor a synthetic polymer graft. In some instances, the compositionincludes an anti-bacterial or anti-bacterial agents could be included.Methods herein can comprise delivering the composition as a wound repairdevice. In one instance, a composition comprises an alginate bead thatis coated with an ECM composition as described herein.

In some instances, the composition is injectable. An injectablecomposition can be, without limitation, a powder, liquid, particles,fragments, gel, or emulsion. The injectable composition can be injectedinto an injured tissue or organ. The compositions herein can recruit,for example without limitation, endothelial, smooth muscle, skeletalmuscle, progenitors, and stem cells.

The composition of the present invention can be developed for substratecoating for a variety of applications. In some instances, the ECM of thecomposition retains a complex mixture of muscle-specific ECM componentsafter solubilization. In some instances, the coatings herein can moreappropriately emulate the native muscle ECM in vitro.

In some instances, a composition herein is a coating. The coating cancomprise an ECM from any tissue for example cardiac muscle, skeletalmuscle, pericardium, liver, adipose tissue, and brain. A coating can beused for tissue culture applications, both research and clinical. Thecoating can be used to coat, for example without limitation, syntheticor other biologic scaffolds/materials, or implants. In some instances, acoating is texturized or patterned. In some instances, a method ofmaking a coating includes adsorption or chemical linking. A thin gel oradsorbed coating can be formed using an ECM solution form of thecomposition. In some instances, a composition herein is configured toseal holes in the heart such as septal defects. The compositions of thepresent invention may be used as coating for biologics, medical devicesor drug delivery devices.

The native ECM is a complex combination of fibrous proteins andproteoglycans that can affect many aspects of cellular behavior. Toregenerate tissue, a scaffold should mimic this native microenvironment.The present invention, therefore, provides an injectable hydrogelderived from skeletal muscle ECM, which mimics the native biochemicalcues, as well as being amenable to minimally invasive, injectableprocedures, providing an advantage for treating PAD and CLI. In certainembodiments, the invention can be used as a delivery vehicle combinedwith cells and/or growth factors. In certain embodiments of theinvention, the compositions and methods herein provide skeletal muscleECM material as an acellular stand-alone therapy, which is used torecruit endogenous cells for neovascularization and repair. In certainembodiments, the present invention provides a porcine source of skeletalmuscle matrix. Xenogeneic decellularized extracellular matrices arebiocompatible upon removal of the cellular antigens, and can be utilizedin the clinic for a number of surgical repair applications.

A liquid version of skeletal muscle matrix herein can form a porousscaffold upon injection, which promotes cellular infiltration to thedamaged area. In certain embodiments, remnant growth factors arepresent. In other embodiments, remnant growth factors are not present.In methods herein, the decellularization and subsequent processing intothe hydrogel form decreases the probability of the presence of remnantgrowth factors.

In the present invention, mitogenic properties of the degradationproducts of the skeletal muscle ECM material were assessed on smoothmuscle cells, a relevant cell type for vascularization. The skeletalmuscle matrix degradation fragments induced a higher proliferation ratecompared to collagen. Extracellular matrix degradation productssometimes have mitogenic activity. The examples herein provide evidencethat the injectable skeletal muscle matrix scaffold inducesneovascularization in vivo.

Therefore, the ability of this present scaffold to induceneovascularization was then assessed in a rat hindlimb ischemia modelcompared to collagen, which is the predominant component of the skeletalmuscle matrix and a commonly utilized scaffold. Not only was the vesseldensity higher in the skeletal muscle matrix, but there weresignificantly more large-diameter vessels greater than 25 μm, indicatingmaturation of the vasculature. Additionally, significance was seen asearly as three days post-injection demonstrating the fast rate ofvascularization. In PAD, the formation of new blood vessels is criticalto treat the ischemic tissue. The presence of more mature vasculatureindicates permanence of the formed vessels, which is important togetting a vascular supply as quickly as possible to the ischemic region,and to maintain blood flow (Banker and Goslin, 1998).

In addition to treating the ischemic environment, increasing muscle massis beneficial in a therapy for PAD and CLI, as patients often sufferwith muscle fatigue and atrophy. In certain embodiments, the liquid formof decellularized skeletal muscle, when utilized as a coating for cellculture, increased skeletal myoblast differentiation compared tocollagen coatings. In certain embodiments, the composition providestissue specific biochemical cues to recapitulate the skeletal musclemicroenvironment. The invention demonstrated that the degradationproducts of this scaffold increased myoblast proliferation compared tocollagen, which is consistent with literature demonstrating theinhibitory effect of collagen on smooth muscle cells and fibroblasts(Koyama et al., 1996; Rhudy and McPherson, 1988).

Next, the infiltration of muscle cells into the scaffold in the hindlimbischemia model was assessed. The number of desmin- and MyoD-positivecells that were recruited into the skeletal muscle matrix scaffold wasmeasured as compared to that into the collagen. Desmin, a musclespecific protein, confirms that the cells that infiltrated were from amyogenic origin. MyoD, on the other hand, is a specific striated muscleregulatory transcription factor, which coordinates the myogenic programin differentiating myoblasts (Kanisicak et al., 2009; Lee et al., 2000;Wada et al., 2002). The invention provides that there were asignificantly higher number of muscle cell types in the skeletal musclematrix, and that many of these cells were also proliferating. TheMyoD-positive cells also indicate that immature progenitor cell typesare recruited to the skeletal muscle matrix. The presence of theseMyoD-positive and desmin-positive muscle cells indicate that theskeletal muscle scaffold is recruiting relevant cell types that aid inthe regeneration of the damaged muscle, in addition to treating theischemic tissue.

The present invention, thus, provides an acellular, biomaterial-onlytherapy for treating PAD. Previous biomaterial strategies for PAD haveonly utilized scaffolds to enhance cell or growth factor therapy (Doi etal., 2007; Jay et al., 2008; Kong et al., 2008; Layman et al., 2007; Leeet al., 2010; Ruvinov et al., 2010; Silva and Mooney, 2007). To increaseits therapeutic benefit, the invention can be used in conjunction withcell or growth factor therapy as these components can be added to thebiomaterial prior to injection. To create a material that could beeasily prepared in the clinic, a method that allowed for long-termstorage of the injectable skeletal muscle matrix scaffold was alsodeveloped with only sterile water required to resuspend it immediatelyprior to use.

The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. It is apparent for skilled artisans that variousmodifications and changes are possible and are contemplated within thescope of the current invention.

EXAMPLES Example 1 Injectable Skeletal Muscle Matrix

Skeletal muscle matrix material was derived through decellularization ofporcine skeletal muscle tissue (FIG. 1A). Fat and connective tissue wasremoved, and the skeletal muscle was cut into ˜1 cm³ pieces, rinsed withdeioninized water and stirred in 1% (wt/vol) solution of SDS in PBS for4-5 days. The decellularized muscle was then stirred overnight indeionized water, and agitated rinses under running deionized water wereperformed to remove residual SDS. In addition to confirmation with lackof nuclei on H&E stained sections (not shown), the DNA content of thematerial was measured as 26.14±1.67 ng of DNA/mg of dry weight ECM,which confirmed decellularization. The matrix was then lyophilized (FIG.1B) and milled into a fine particulate. At this stage the material canbe hydrated and utilized for in vivo injection or it can beenzymatically digested to form a liquid (FIG. 1C). At this stage, theliquid skeletal muscle matrix can be diluted and utilized as a coatingfor cell culture, or can be brought to physiological pH and temperature,which triggers assembly into a hydrogel (FIG. 1D). After raising the pHof the material to 7.4 at room temperature, the material can also bere-lyophilized (FIG. 1E) for long-term storage at −80° C. The materialcan then be resuspended at a later date using only sterile water (FIG.1F) and utilized for in vivo injection.

Example 2 Mitogenic Assay

Degradation products of decellularized ECMs have been previously shownto have mitogenic activity. It was examined whether the degradationproducts of the skeletal muscle matrix hydrogel had a mitogenic effecton cells in vitro. Proliferation of smooth muscle cells and skeletalmyoblasts following exposure to either enzymatically degraded skeletalmuscle ECM or collagen was assessed. Pepsin was also included as acontrol, as pepsin was utilized to digest the matrix material. APicogreen assay was used to determine double stranded DNA content atdays 3, 5, and 7 in culture to quantify cell proliferation. It was foundthat both smooth muscle cells (FIG. 2A) and myoblasts (FIG. 2B), whencultured in media containing degraded skeletal muscle matrix, had ahigher rate of proliferation compared to cells cultured in mediacontaining the same concentration of collagen. The increase in cellnumber was significantly greater at all time points (p<0.01). At day 3,there was a 1.85-fold increase in cell number in the skeletal musclematrix wells compared to collagen for the smooth muscle cells, and acorresponding 2.15-fold increase with the skeletal myoblasts. There wasalso a 1.3 fold increase for skeletal muscle matrix wells compared topepsin for both cell types, while the pepsin and collagen controls werenot statistically different. Thus, degradation products of the skeletalmuscle matrix were shown to promote mitogenic activity in both celltypes in vitro when compared to collagen or the pepsin control.

Example 3 Gelation In Vitro and In Vivo

A gel form of the matrix was initially made in vitro by bringing thematerial (6 mg/mL) to a physiological pH and incubating the material at37° C. After gelation, the material was tested for rheologicalproperties where it was determined that the material had a storagemodulus (G′) of 6.5±0.5 Pa. A representative trace of rheological datais shown in FIG. 3. The ability of the liquid skeletal muscle matrix toform a gel in situ were then assessed by injecting the material into ahealthy rat hindlimb. For all in vivo studies, liquid skeletal musclematrix, which had been biotinylated and re-lyophilized for storage at−80 C were utilized. Prior to injection, the material was resuspended insterile water alone. The skeletal muscle matrix was then loaded into asyringe and injected intramuscularly into a rat hindlimb (FIG. 4A). Todetermine whether the skeletal muscle matrix would assemble and form ascaffold, the injection region was excised after 20 minutes. A visiblegel, denoted by the white region in FIG. 4B, was observed within themuscle. Additional matrix injections were cryosectioned and stained tovisualize the biotinylated matrix. The liquid skeletal muscle matrixassembled into a fibrous scaffold once in vivo (FIG. 4C). To assess themicroarchitecture of the skeletal muscle matrix hydrogel, the materialwas injected subcutaneously, and excised after 20 minutes. ScanningElectron Microscopy (SEM) demonstrated that the matrix forms a porous,fibrous scaffold, both in vitro and in vivo, that is composed of fiberson the nano- and micro-scale (FIG. 5).

Example 4 Cellular Infiltration and Neovascularization

Upon confirmation that the material was able to assemble upon injection,the skeletal muscle matrix hydrogel were then examined in a rat hindlimbischemia model to assess its potential for treating PAD. One weekpost-hindlimb ischemia, either skeletal muscle matrix or collagen wasinjected intramuscularly below the site of femoral artery resection. At3, 5, 7 or 14 days post-injection, the muscle was harvested to determinecellular infiltration. The hydrogel was still present at all timepoints, although it had significantly degraded by day 14. At each timepoint, the amount of neovascularization, which would be critical totreat the ischemic tissue, as well as the number of muscle cells andmuscle progenitors, which could aid in repair of the damaged tissue,were assessed.

To determine whether the acellular scaffold would support new vesselformation in vivo, smooth muscle cells in collagen (FIG. 6A) andskeletal muscle matrix (FIG. 6B) injected regions were labeled viaimmunohistochemistry. Arteriole density was significantly greater in theskeletal muscle matrix injection region compared to collagen at 3, 5,and 7 days post-injection (FIG. 6C), with many of the vessels having anaverage diameter greater than 25 μm (FIG. 6D). While not significant,there was still a distinct trend towards an increase in vasculature atday 14 following injection of the skeletal muscle matrix hydrogel.Additionally, endothelial cell infiltration was measured in collagen(FIG. 7A), and skeletal muscle matrix (FIG. 7B) injection regions.Endothelial cell density was found to be similar across all four timepoints, but was significantly greater in the skeletal muscle matrixinjection region at 3 and 7 days post-injection (FIG. 7C).

In PAD and CLI, patients often suffer from general muscle fatigue andatrophy, and therefore, in addition to increasing vascularization,restoring muscle mass would also be beneficial. It was then determinedwhether muscle cells were also recruited to the injection site usingstaining against desmin (FIGS. 8A,B). The desmin positive cells werealso co-stained for Ki67, a marker for proliferation, as denoted by thearrows in FIG. 8. The skeletal muscle matrix recruited significantlymore desmin-positive cells when compared to the collagen matrix at 3, 5,and 7 days post-injection, a the trend that continued at day 14 (FIG.8C). Moreover, the majority of cells expressing desmin also were Ki67positive, indicating proliferating muscle cells were infiltrating theinjection region (FIG. 8D). The number of Ki67 and desmin positive cellswas significantly increased at 3, 5, and 7 days post-injection, with thesame trend at day 14. Cell infiltration was further assessed using MyoDas a marker for the potential recruitment of activated satellite cells.There was a low number of MyoD positive cells recruited into theinjection region of both materials at the examined time points; however,there was a statistically significant increase in MyoD positive cells inthe skeletal muscle matrix (FIG. 9). The MyoD staining was prevalentlyperinuclear, which has been shown in other studies.

Example 5 Injectable Skeletal Muscle Matrix Powder

After milling, the skeletal muscle matrix powder is hydrated withsterile water, saline, or PBS and injected or implanted into the injuredlimb. Depending on the concentration, the injected skeletal musclematrix can form a bolus or spread throughout the tissue. Uponimplantation, the skeletal muscle ECM particulate forms a scaffold andcreates degradation products that recruit endogenous cells to repair theischemic region. These cells include blood vessels, skeletal musclecells, and skeletal muscle progenitors. By recruiting endogenous cellsfor repair and regeneration, the skeletal muscle matrix particulate hasto potential to treat peripheral artery disease and critical limbischemia, and the various complications associated with these diseases.

Example 6 Materials, Methods and Discussion Decellularization ofSkeletal Muscle for Matrix Preparation

Skeletal muscle from the hindleg was harvested from Yorkshire pigs,approximately 30-45 kg, immediately after sedation with aketamine/xylazine combination (25 mg/kg, 2 mg/kg respectively) andeuthanasia with beuthanasia (1 mL/5 kg). Fat and connective tissue wasremoved, and the skeletal muscle was cut into ˜1 cm³ pieces anddecellularized. Briefly, the tissue was rinsed with deionized water andstirred in 1% (wt/vol) solution of sodium dodecyl sulfate (SDS) inphosphate buffered saline (PBS) for 4-5 days. Decellularized skeletalmuscle was stirred overnight in deionized water and then agitated rinsesunder running DI water were performed to remove residual SDS. A sampleof decellularized matrix was frozen in Tissue Tek O.C.T. freezingmedium, sectioned into 10 μm slices, and stained with hematoxylin andeosin (H&E) to confirm the absence of nuclei. Following thedecellularization protocol, the ECM was lyophilized overnight and milledto a fine powder using a Wiley Mini Mill. Additionally, to quantify DNAcontent, the DNeasy assay (Qiagen, Valencia, Calif.) was performedaccording to manufacturer's instructions. After extraction, the Take3plate was used to measure the concentration of DNA using a Synergy 2microplate reader (Biotek, Winooski, Vt.).

Preparation of Injectable Skeletal Muscle Matrix and Collagen

In order to render the decellularized extracellular matrix (ECM) into aliquid form, the milled form of the matrix was subjected to enzymaticdigestion. Pepsin (SIGMA, St. Louis, Mo.) was dissolved in 0.1 Mhydrochloric acid (HCl) to make a 1 mg/ml pepsin solution and thenfiltered through a 0.22 μm filter (Millipore, Billerca, Mass.). The ECMat a ratio of 10:1 was digested in the pepsin solution under constantstirring. After approximately 48 hours, the matrix was brought to aphysiological pH in a BSL-2 safety cabinet, and then either diluted forin vitro assays or for injection. For in vitro and in vivo studies, theskeletal muscle matrix was brought to a pH of 7.4 through the additionof sterile-filtered sodium hydroxide (NaOH) and 10×PBS, and furtherdiluted to 6 mg/ml using 1×PBS inside a BSL-2 safety cabinet.

Skeletal Muscle Matrix In Vitro Gel Characterization

Gels of the skeletal muscle matrix were formed, at a concentration 6mg/ml for rheological characteristics and for scanning electronmicroscopy. Either 100 μl of matrix was pipetted into a 96 well plate(Corning, Corning, N.Y.) or 500 μl in glass scintillation vials andincubated overnight to form gels. Rheometry was conducted on the 500 μlin vitro-formed skeletal muscle matrix gels using a TA instruments AR-G2rheometer. The gels were tested using a 20 mm parallel plate geometrywith a 1.2 mm gap at 37° C. Three frequency sweeps were performed withinthe linear viscoelastic strain region. Samples were run in triplicateand then the values were averaged to calculate the storage modulus.

Scanning electron microscopy (SEM) was utilized to determine themicrostructure of the skeletal muscle matrix gels. These gels wereeither formed in vivo by injecting the skeletal muscle matrixsubcutaneously in a rat and excised after 20 minutes, or in vitro afterincubation of the material in a 96 well plate at 37° C. overnight. Theskeletal muscle matrix gels were harvested and fixed with 2.5%glutaraldehyde for 2 hours, and then dehydrated using a series ofethanol rinses (30-100%). Samples were then critical point dried andcoated with iridium using an Emitech K575X Sputter coater. Electronmicroscopy images were taken using a Phillips XL30 Environmental SEMField Emission microscope at 10 kV, with 242 μA and a working distanceof 10 mm.

In Vitro Proliferation Assays

Primary rat aortic smooth muscle cells (RASMC) and C2C12 skeletalmyoblasts were maintained on collagen coated plates and split at 1:5every 2-3 days. Cells between passages 4 and 10 were plated at 750cells/well in 96 well plates in growth media consisting of DMEM, 10%fetal bovine serum, and 1% pen-strep solution. Twenty-four hours later,the cells were washed with PBS to remove non-adherent cells. Digestedskeletal muscle matrix and collagen were brought to a pH of 7.4, andthen added to the growth media at concentrations of 0.05 mg/mL. As theECM was enzymatically digested, pepsin was also included as a control at0.005 mg/mL. All conditions were run in quadruplicate. Every two days,media was changed and cell proliferation was assessed using thePICOGREEN® assay (Invitrogen) per manufacturer's directions. Briefly,wells were rinsed in PBS and then incubated with 100 μL of TE buffer.After incubation for 30 minutes at room temperature followed by 5minutes on a shaker, 100 μL of 1:200 Picogreen reagent was added. Uponcovering the plates in foil and shaking them for 30 minutes, doublestranded DNA was quantified using a fluorescent plate reader at 630 nmat days 3, 5, and 7.

In Vivo Gelation Test

To prepare for in vivo studies, a preliminary test was performed toensure that the skeletal muscle matrix would be able to gel uponinjection. The skeletal muscle matrix was labeled with biotin, and theninjected into the hindlimbs of healthy Sprague Dawley rats. For biotinlabeling, a 10 mM solution of EZ link Sulfo-NHS-Biotin (Pierce,Rockford, Ill.) was prepared and mixed with the liquid skeletal musclematrix for a final concentration of 0.3 mg of biotin/mg matrix. Themixture was allowed to sit on ice for two hours. The skeletal musclematrix was then frozen, lyophilized and stored at −80° C. until use. Toresuspend the skeletal muscle matrix, sterile water was added at theoriginal volume to bring the material to 6 mg/ml and vortexed. FemaleHarlan Sprague Dawley rats (225-250 g) were anesthetized usingisoflurane at 5%, intubated, and maintained at 2.5% isoflurane duringsurgery. In preliminary studies, (n=2) 150 μl of skeletal muscle matrixwas injected intramuscularly into healthy rats. The muscle was excisedafter 20 min, and fresh frozen using Tissue Tek O.C.T.

Hindlimb Ischemia Model

After confirmation of gelation in vivo, a rat hindlimb ischemia modelwas utilized to test the skeletal muscle extracellular matrix. Animalswere placed in a supine position and hindlimb ischemia was induced byligation and excision of the femoral artery. After ligation of theproximal end of the femoral artery, the distal portion of the saphenousartery was ligated and the artery and side branches were dissected free,and then excised. The area was sutured closed and animals were given ananalgesic of 0.05 mg/kg of buprenorphine hydrochloride (ReckittBenckiser Healthcare (UK) Ltd., Hull, England) prior to recovery fromanesthesia. One week post-injury, the rats were anesthetized using 5%isoflurane, intubated, and maintained at 2.5% isoflurane for injection.Skeletal muscle matrix and rat tail collagen were biotinylated in orderto visualize the injection region and 150 μl was injectedintramuscularly. Injection was confirmed by a lightening of the muscleat the site of injection. Rats were sacrificed using an overdose ofsodium pentobarbital (200 mg/kg) at 3, 5, 7, or 14 days post injection(n=4, except n=3 for 14 day collagen injection), and leg muscles wereharvested and frozen in Tissue Tek O.C.T.

Histology and Immunohistochemistry

The excised muscle was cryosectioned into 10 μm slices. Slices werestained with Hematoxylin and Eosin every 1 mm and screened to determinethe location of injected material. Adjacent slides were stained forvisualization of biotin-labeled skeletal muscle matrix or collagen, toconfirm the injection site. Slides were fixed in acetone, incubated withsuperblock buffer (Pierce), followed by 3% hydrogen peroxide (Sigma),and horseradish peroxidase conjugated neutravidin (Pierce) at roomtemperature. The reaction was visualized by incubation withdiaminobenzidine (DAB, Pierce) for ten minutes.

Five slides evenly spaced within the injection region were then used forimmunohistochemistry (IHC). Sections were fixed for 2 min in acetone andblocked with staining buffer for 1 h (2% goat serum and 0.3% TritonX-100 in PBS). Skeletal muscle sections were then assessed for vesselformation using a mouse anti-smooth muscle actin antibody (Dako,Carpinteria, Calif.; 1:75 dilution) to label smooth muscle cells. Afterthree 5-minute washes with PBS, AlexaFluor 568 anti-mouse (Invitrogen,1:200 dilution) was used as a secondary. Endothelial cell infiltrationwas assessed using FITC labeled isolectin (Vector Laboratories,Burlingame, Calif.; 1:100 dilution). Slides were then mounted usingFluoromount (Sigma). Sections stained with only the primary antibody orsecondary antibody were used as negative controls. Images were taken at100× using Carl Zeiss Observer D.1 and analyzed using AxioVisionsoftware. Arterioles were quantified with a visible lumen and adiameter≧10 μm and normalized over the injection area.

In order to assess proliferating muscle cell infiltration into theinjection region, sections were stained using a mouse anti-desminantibody (Sigma; dilution 1:100) and co-stained with a rabbit anti-Ki67(Santa Cruz Biotech, Santa Cruz, Calif.; dilution 1:100). AlexaFluor 488anti-mouse and AlexaFluor 568 anti-rabbit were used for secondaryantibodies (1:200), followed by staining with Hoechst 33342. Slides weremounted with Fluoromount (Sigma) prior to imaging. Additionally, theskeletal muscle tissue was assessed using a rabbit anti-MyoD (Santa CruzBiotech, Santa Cruz, Calif.; dilution 1:100), followed by AlexaFluor 488anti-rabbit as a secondary antibody, and Hoechst 33342. Three 400×images were taken per slide and analyzed using AxioVision software. Thenumber of desmin positive cells, and desmin positive cells thatco-localized with Ki67 were counted, averaged and normalized over thearea. For the tissue sections analyzed for MyoD, the number of positivecells with MyoD co-localized with nuclei were counted and averaged overthe area of injection.

Statistical Analysis

All data is presented as the mean±standard error of mean. For the invitro assays, samples were run in quadruplicate and results wereaveraged. Significance was determined using a one-way analysis ofvariance (ANOVA) with a Bonferroni post-test. A two-tailed Student'st-test was used for all other data and reported as p<0.05 and p<0.001.

In conclusion, the present invention provides an injectable skeletalmuscle extracellular matrix scaffold as a new biomaterial-only therapyfor treating PAD and CLI and complications associated with thesediseases. The invention demonstrates that decellularized skeletal muscleECM can be processed to form an injectable matrix material, whichassembles into a porous and fibrous scaffold in vivo. The invention alsodemonstrates that degradation products of the material induceproliferation of smooth muscle cells and skeletal myoblasts in vitro.Furthermore, the invention provides that the injected scaffold increasedneovascularization and infiltration of muscle cells within thebiomaterial as compared to collagen, suggesting that it improvesneovascularization in PAD, as well as treat the associated muscleatrophy. Thus, the invention provides a novel therapeutic treatmentmethod for PAD and CLI with an injectable scaffold derived fromdecellularized skeletal muscle ECM.

REFERENCES

-   1. Alev C, Ii M, Asahara T (2011) Endothelial progenitor cells: a    novel tool for the therapy of ischemic diseases. Antioxid Redox    Signal 15: 949-965.-   2. Bach A D, Arkudas A, Tjiawi J, Polykandriotis E, Kneser U, Horch    R E, Beier J P (2006) A new approach to tissue engineering of    vascularized skeletal muscle. J Cell Mol Med 10: 716-726.-   3. Badylak S F (2007) The extracellular matrix as a biologic    scaffold material. Biomaterials 28: 3587-3593.-   4. Badylak S F, Freytes D O, Gilbert T W (2009) Extracellular matrix    as a biological scaffold material: Structure and function. Acta    Biomater 5: 1-13.-   5. Badylak S F, Gilbert T W (2008) Immune response to biologic    scaffold materials. Semin Immunol 20: 109-116.-   6. Badylak S F, Park K, Peppas N, McCabe G, Yoder M (2001)    Marrow-derived cells populate scaffolds composed of xenogeneic    extracellular matrix. Exp Hematol 29: 1310-1318.-   7. Banker G, Goslin K (1998) Culturing Nerve Cells. The MIT Press.-   8. Beattie A J, Gilbert T W, Guyot J P, Yates A J, Badylak S    F (2008) Chemoattraction of Progenitor Cells by Remodeling    Extracellular Matrix Scaffolds. Tissue Eng Part A 15: 1119-1125.-   9. Belch J J, Topol E J, Agnelli G, Bertrand M, Califf R M, Clement    D L, Creager M A, Easton J D, Gavin J R, 3rd, Greenland P, Hankey G,    Hanrath P, Hirsch A T, Meyer J, Smith S C, Sullivan F, Weber M    A (2003) Critical issues in peripheral arterial disease detection    and management: a call to action. Arch Intern Med 163: 884-892.-   10. Bhang S H, Kim J H, Yang H S, La W G, Lee T J, Kim G H, Kim H A,    Lee M, Kim B S (2011) Combined gene therapy with hypoxia-inducible    factor-1alpha and heme oxygenase-1 for therapeutic angiogenesis.    Tissue engineering. Part A 17: 915-926.-   11. Bruey J M, Kantarjian H, Ma W, Estrov Z, Yeh C, Donahue A,    Sanders H, O'Brien S, Keating M, Albitar M (2010) Circulating Ki-67    index in plasma as a biomarker and prognostic indicator in chronic    lymphocytic leukemia. Leuk Res 34: 1320-1324.-   12. Chan Y C, Cheng S W (2011) Drug-eluting stents and balloons in    peripheral arterial disease: evidence so far. Int J Clin Pract 65:    664-668.-   13. Christman K L, Vardanian A J, Fang Q, Sievers R E, Fok H H, Lee    R J (2004) Injectable fibrin scaffold improves cell transplant    survival, reduces infarct expansion, and induces neovasculature    formation in ischemic myocardium. J Am Coll Cardiol 44: 654-660.-   14. Cooper R N, Tajbakhsh S, Mouly V, Cossu G, Buckingham M,    Butler-Browne G S (1999) In vivo satellite cell activation via Myf5    and MyoD in regenerating mouse skeletal muscle. J Cell Sci 112 (Pt    17): 2895-2901.-   15. Crapo P M, Gilbert T W, Badylak S F (2011) An overview of tissue    and whole organ decellularization processes. Biomaterials 32:    3233-3243.-   16. Dattilo P B, Casserly I P (2011) Critical limb ischemia:    endovascular strategies for limb salvage. Prog Cardiovasc Dis 54:    47-60.-   17. DeQuach J A, Mezzano V, Miglani A, Lange S, Keller G M, Sheikh    F, Christman K L (2010) Simple and high yielding method for    preparing tissue specific extracellular matrix coatings for cell    culture. PLoS One 5: e13039.-   18. Diniz G, Aktas S, Turedi A, Temir G, Ortac R, Vergin C (2011)    Telomerase reverse transcriptase catalytic subunit expression and    proliferation index in Wilms tumor. Tumour Biol 32: 761-767.-   19. Doi K, Ikeda T, Marui A, Kushibiki T, Arai Y, Hirose K, Soga Y,    Iwakura A, Ueyama K, Yamahara K, Itoh H, Nishimura K, Tabata Y,    Komeda M (2007) Enhanced angiogenesis by gelatin hydrogels    incorporating basic fibroblast growth factor in rabbit model of hind    limb ischemia. Heart Vessels 22: 104-108.-   20. Fadini G P, Agostini C, Avogaro A (2010) Autologous stem cell    therapy for peripheral arterial disease meta-analysis and systematic    review of the literature. Atherosclerosis 209: 10-17.-   21. Gilbert T W, Sellaro T L, Badylak S F (2006) Decellularization    of tissues and organs. Biomaterials 27: 3675-3683.-   22. Gupta R, Losordo D W (2011) Cell therapy for critical limb    ischemia: moving forward one step at a time. Circ Cardiovasc Interv    4: 2-5.-   23. Hidestrand M, Richards-Malcolm S, Gurley C M, Nolen G, Grimes B,    Waterstrat A, Zant G V, Peterson C A (2008) Sca-1-expressing    nonmyogenic cells contribute to fibrosis in aged skeletal muscle. J    Gerontol A Biol Sci Med Sci 63: 566-579.-   24. Jay S M, Shepherd B R, Bertram J P, Pober J S, Saltzman W    M (2008) Engineering of multifunctional gels integrating highly    efficient growth factor delivery with endothelial cell    transplantation. Faseb J 22: 2949-2956.-   25. Jeon O, Krebs M, Alsberg E (2011) Controlled and sustained gene    delivery from injectable, porous PLGA scaffolds. J Biomed Mater Res    A 98: 72-79.-   26. Kanisicak O, Mendez J J, Yamamoto S, Yamamoto M, Goldhamer D    J (2009) Progenitors of skeletal muscle satellite cells express the    muscle determination gene, MyoD. Developmental biology 332: 131-141.-   27. Kawamoto A, Katayama M, Handa N, Kinoshita M, Takano H, Horii M,    Sadamoto K, Yokoyama A, Yamanaka T, Onodera R, Kuroda A, Baba R,    Kaneko Y, Tsukie T, Kurimoto Y, Okada Y, Kihara Y, Morioka S,    Fukushima M, Asahara T (2009) Intramuscular transplantation of    G-CSF-mobilized C D34(+) cells in patients with critical limb    ischemia: a phase I/IIa, multicenter, single-blinded,    dose-escalation clinical trial. Stem Cells 27: 2857-2864.-   28. Kong H J, Kim E S, Huang Y C, Mooney D J (2008) Design of    biodegradable hydrogel for the local and sustained delivery of    angiogenic plasmid DNA. Pharm Res 25: 1230-1238.-   29. Koyama H, Raines E W, Bornfeldt K E, Roberts J M, Ross R (1996)    Fibrillar collagen inhibits arterial smooth muscle proliferation    through regulation of Cdk2 inhibitors. Cell 87: 1069-1078.-   30. Kuraitis D, Zhang P, Zhang Y, Padavan D T, McEwan K, Sofrenovic    T, McKee D, Zhang J, Griffith M, Cao X, Musaro A, Ruel M, Suuronen E    J (2011) A stromal cell-derived factor-1 releasing matrix enhances    the progenitor cell response and blood vessel growth in ischaemic    skeletal muscle. Eur Cell Mater 22: 109-123.-   31. Lawall H, Bramlage P, Amann B (2010) Stem cell and progenitor    cell therapy in peripheral artery disease. A critical appraisal.    Thromb Haemost 103: 696-709.-   32. Layman H, Rahnemai-Azar A A, Pham S M, Tsechpenakis G,    Andreopoulos F M (2011) Synergistic angiogenic effect of    codelivering fibroblast growth factor 2 and granulocyte-colony    stimulating factor from fibrin scaffolds and bone marrow    transplantation in critical limb ischemia. Tissue Eng Part A 17:    243-254.-   33. Layman H, Spiga M G, Brooks T, Pham S, Webster K A, Andreopoulos    F M (2007) The effect of the controlled release of basic fibroblast    growth factor from ionic gelatin-based hydrogels on angiogenesis in    a murine critical limb ischemic model. Biomaterials 28: 2646-2654.-   34. Lee J, Bhang S H, Park H, Kim B S, Lee K Y (2010) Active blood    vessel formation in the ischemic hindlimb mouse model using a    microsphere/hydrogel combination system. Pharm Res 27: 767-774.-   35. Lee J Y, Qu-Petersen Z, Cao B, Kimura S, Jankowski R, Cummins J,    Usas A, Gates C, Robbins P, Wernig A, Huard J (2000) Clonal    isolation of muscle-derived cells capable of enhancing muscle    regeneration and bone healing. J Cell Biol 150: 1085-1100.-   36. Li F, Li W, Johnson S, Ingram D, Yoder M, Badylak S (2004)    Low-molecular-weight peptides derived from extracellular matrix as    chemoattractants for primary endothelial cells. Endothelium 11:    199-206.-   37. Lutolf M P, Hubbell J A (2005) Synthetic biomaterials as    instructive extracellular microenvironments for morphogenesis in    tissue engineering. Nat Biotechnol 23: 47-55.-   38. Manzi M, Palena L, Cester G (2011) Endovascular techniques for    limb salvage in diabetics with crural and pedal disease. J    Cardiovasc Surg (Torino) 52: 485-492.-   39. Megeney L A, Kablar B, Garrett K, Anderson J E, Rudnicki M    A (1996) MyoD is required for myogenic stem cell function in adult    skeletal muscle. Genes Dev 10: 1173-1183.-   40. Menasche P (2010) Cell therapy for peripheral arterial disease.    Curr Opin Mol Ther 12: 538-545.-   41. Merritt E K, Hammers D W, Tierney M, Suggs L J, Walters T J,    Farrar R P Functional assessment of skeletal muscle regeneration    utilizing homologous extracellular matrix as scaffolding. Tissue Eng    Part A 16: 1395-1405.-   42. Numata S, Fujisato T, Niwaya K, Ishibashi-Ueda H, Nakatani T,    Kitamura S (2004) Immunological and histological evaluation of    decellularized allograft in a pig model: comparison with    cryopreserved allograft. J Heart Valve Dis 13: 984-990.-   43. Ott H C, Matthiesen T S, Goh S K, Black L D, Kren S M, Netoff T    I, Taylor D A (2008) Perfusion-decellularized matrix: using nature's    platform to engineer a bioartificial heart. Nat Med 14: 213-221.-   44. Reing J E, Zhang L, Myers-Irvin J, Cordero K E, Freytes D O,    Heber-Katz E, Bedelbaeva K, McIntosh D, Dewilde A, Braunhut S J,    Badylak S F (2009) Degradation products of extracellular matrix    affect cell migration and proliferation. Tissue Eng Part A 15:    605-614.-   45. Rhudy R W, McPherson J M (1988) Influence of the extracellular    matrix on the proliferative response of human skin fibroblasts to    serum and purified platelet-derived growth factor. J Cell Physiol    137: 185-191.-   46. Rieder E, Nigisch A, Dekan B, Kasimir M T, Muhlbacher F, Wolner    E, Simon P, Weigel G (2006) Granulocyte-based immune response    against decellularized or glutaraldehyde cross-linked vascular    tissue. Biomaterials 27: 5634-5642.-   47. Ruvinov E, Leor J, Cohen S (2010) The effects of controlled HGF    delivery from an affinity-binding alginate biomaterial on    angiogenesis and blood perfusion in a hindlimb ischemia model.    Biomaterials 31: 4573-4582.-   48. Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known    and the unknown. J Cell Physiol 182: 311-322.-   49. Seif-Naraghi S B, Salvatore M A, Schup-Magoffin P J, Hu D P,    Christman K L (2010) Design and characterization of an injectable    pericardial matrix gel: a potentially autologous scaffold for    cardiac tissue engineering. Tissue Eng Part A 16: 2017-2027.-   50. Silva E A, Mooney D J (2007) Spatiotemporal control of vascular    endothelial growth factor delivery from injectable hydrogels    enhances angiogenesis. J Thromb Haemost 5: 590-598.-   51. Singelyn J M, DeQuach J A, Seif-Naraghi S B, Littlefield R B,    Schup-Magoffin P J, Christman K L (2009) Naturally derived    myocardial matrix as an injectable scaffold for cardiac tissue    engineering. Biomaterials 30: 5409-5416.-   52. Singelyn J M, Sundaramurthy P, Johnson T D, Schup-Magoffin P J,    Hu D P, Faulk D M, Wang J, Mayle K M, Bartels K, Salvatore M, Kinsey    A M, Demaria A N, Dib N,-   Christman K L (2012) Catheter-deliverable hydrogel derived from    decellularized ventricular extracellular matrix increases endogenous    cardiomyocytes and preserves cardiac function post-myocardial    infarction. Journal of the American College of Cardiology 59:    751-763.-   53. Sprengers R W, Lips D J, Moll F L, Verhaar M C (2008) Progenitor    cell therapy in patients with critical limb ischemia without    surgical options. Ann Surg 247: 411-420.-   54. Stansby G, Williams R (2011) Angioplasty for treatment of    isolated below-the-knee arterial stenosis in patients with critical    limb ischemia. Angiology 62: 357-358.-   55. Sundararaghavan H G, Metter R B, Burdick J A (2010) Electrospun    fibrous scaffolds with multiscale and photopatterned porosity.    Macromol Biosci 10: 265-270.-   56. Tongers J, Roncalli J G, Losordo D W (2008) Therapeutic    angiogenesis for critical limb ischemia: microvascular therapies    coming of age. Circulation 118: 9-16.-   57. Uriel S, Labay E, Francis-Sedlak M, Moya M L, Weichselbaum R R,    Ervin N, Cankova Z, Brey E M (2009) Extraction and assembly of    tissue-derived gels for cell culture and tissue engineering. Tissue    Eng Part C Methods 15: 309-321.-   58. Uygun B E, Soto-Gutierrez A, Yagi H, Izamis M L, Guzzardi M A,    Shulman C, Milwid J, Kobayashi N, Tilles A, Berthiaume F, Hertl M,    Nahmias Y, Yarmush M L, Uygun K (2010) Organ reengineering through    development of a transplantable recellularized liver graft using    decellularized liver matrix. Nat Med 16: 814-820.-   59. Valentin J E, Turner N J, Gilbert T W, Badylak S F (2010)    Functional skeletal muscle formation with a biologic scaffold.    Biomaterials 31: 7475-7484.-   60. Wada M R, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto    N (2002) Generation of different fates from multipotent muscle stem    cells. Development 129: 2987-2995.-   61. Webber M J, Tongers J, Newcomb C J, Marquardt K T, Bauersachs J,    Losordo D W, Stupp S I (2011) Supramolecular nanostructures that    mimic VEGF as a strategy for ischemic tissue repair. Proceedings of    the National Academy of Sciences of the United States of America    108: 13438-13443.-   62. Wolf M T, Daly K A, Reing J E, Badylak S F (2012) Biologic    scaffold composed of skeletal muscle extracellular matrix.    Biomaterials 33: 2916-2925.-   63. Yamamoto D L, Csikasz R I, Li Y, Sharma G, Hjort K, Karlsson R,    Bengtsson T (2008) Myotube formation on micro-patterned glass:    intracellular organization and protein distribution in C2C12    skeletal muscle cells. J Histochem Cytochem 56: 881-892.-   64. Young D A, Ibrahim D O, Hu D, Christman K L (2011) Injectable    hydrogel scaffold from decellularized human lipoaspirate. Acta    Biomater 7: 1040-1049.-   65. Zantop T, Gilbert T W, Yoder M C, Badylak S F (2006)    Extracellular matrix scaffolds are repopulated by bone    marrow-derived cells in a mouse model of achilles tendon    reconstruction. J Orthop Res 24: 1299-1309.

S While preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method comprising injecting or implanting in a subject withperipheral artery disease an effective amount of a compositioncomprising decellularized extracellular matrix derived from skeletalmuscle tissue.
 2. The method of claim 1, wherein said composition iscoated on an implant.
 3. The method of claim 1, wherein said compositionis delivered as a liquid or a powder.
 4. The method of claim 3, whereinsaid composition transitions to a gel form after delivery.
 5. The methodof claim 1, wherein said composition degrades within one to three monthsfollowing injection or implantation.
 6. The method of claim 1, whereininjection or implantation of said composition repairs damage to skeletalmuscle tissue sustained by said subject.
 7. The method of claim 1,wherein injection or implantation of said composition repairs damagecaused by ischemia in said subject.
 8. The method of claim 1, whereinsaid effective amount is an amount that increases blood flow, increasesmuscle mass, or induces new vascular formation in the area of theinjection or implantation of the subject.
 9. The method of claim 1,wherein the effective amount is effective for treating at least one ofthe symptoms selected from the group consisting of ulcers, traumawounds, draining wounds, inflammation, Buerger disease, atherosclerosisobliterans, thromboangiitis obliterans, and gangrene that is associatedwith peripheral artery disease.
 10. A method comprising injecting orimplanting in a subject with critical limb ischemia an effective amountof a composition comprising decellularized extracellular matrix derivedfrom skeletal muscle tissue.
 11. The method of claim 10, where saidcomposition is coated on an implant.
 12. The method of claim 10, whereinsaid composition is delivered as a liquid or a powder.
 13. The method ofclaim 12, wherein said composition degrades within one to three monthsfollowing injection or implantation.
 14. The method of claim 10, whereininjection or implantation of said composition repairs skeletal muscletissue damage caused by ischemia in said subject.
 15. The method ofclaim 15, wherein said composition transitions to a gel form afterdelivery.
 16. The method of claim 10, wherein said effective amount isan amount that increases blood flow, increases muscle mass, or inducesnew vascular formation in the area of the injection or implantation ofthe treated subject.
 17. The method of claim 10, wherein the effectiveamount is effective for treating at least one of the symptoms selectedfrom the group consisting of ulcers, trauma wounds, draining wounds,inflammation, Buerger disease, atherosclerosis obliterans,thromboangiitis obliterans, and gangrene that is associated withperipheral artery disease.
 18. A method comprising injecting orimplanting in a subject with peripheral artery disease or critical limbischemia an effective amount of a composition comprising decellularizedextracellular matrix derived from a suitable tissue.
 19. The method ofclaim 18, wherein said suitable tissue is selected from the groupconsisting of cardiac, pericardial, liver, brain, small intestinesubmucosa, bladder, lung, and vascular tissue.
 20. The method of claim18, wherein said effective amount is an amount that increases bloodflow, increases muscle mass, or induces new vascular formation in thearea of the injection or implantation of the subject.